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| United States Patent |
5,599,978
|
|
Giannessi
, et al.
|
February 4, 1997
|
Process for manufacturing L-(-)-carnitine from a waste product having
opposite configuration
Abstract
The present invention relates to a proces for manufacturing L-(-)-carnitine
from D-(+)-carnitine or a derivative thereof. D-(+)-carnitine is
esterified in order to protect the carboxyl group and subsequently
converted to an acyl derivative. The acyl derivative is then converted to
a lactone of L-(-)-carnitine. Finally, the lactone is reopened to obtain
L-(-)-carnitine.
| Inventors:
|
Giannessi; Fabio (Rome, IT);
Bolognesi; Maria L. (Bologna, IT);
Tinti; Maria O. (Rome, IT);
De Angelis; Francesco (Rome, IT)
|
| Assignee:
|
Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. (Rome, IT)
|
| Appl. No.:
|
367863 |
| Filed:
|
January 3, 1995 |
Foreign Application Priority Data
| Dec 21, 1992[IT] | RM92A0915 |
| Current U.S. Class: |
562/567 |
| Intern'l Class: |
C07C 229/10; C07C 229/22 |
| Field of Search: |
562/567
|
References Cited
U.S. Patent Documents
| 4413142 | Nov., 1983 | Fiorini et al. | 562/567.
|
Primary Examiner: Trinh; Ba K.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt, P.C.
Parent Case Text
This is a division of application Ser. No. 08/170,764 filed on Dec. 21,
1993 now U.S. Pat. No. 5,412,113.
Claims
What is claimed as new and is desired to be secured by Letters Patent of
the United States is:
1. A process for producing L-(-)-carnitine inner salt from
D-(+)-carnitinamide which comprises:
(a) hydrolyzing a salt of D-(+)-carnitinamide 1 of the general formula
##STR2##
wherein X.sup.- is any monovalent counterion to obtain D-(+)-carnitine 2
##STR3##
(b) esterifying said D-(+)-carnitine 2 to an ester 3 of the general
formula
##STR4##
wherein R.sub.1 is (i) a straight or branched alkoxy group having 1-11
carbon atoms or (ii) an arylalkoxy or diarylalkoxy group wherein the aryl
group is a monocyclic or bicyclic aryl group containing 5 to 12 carbon
atoms and the alkyl group has 1-4 carbon atoms, and wherein said
arylalkoxy or diarylalkoxy groups can be optionally substituted with a
lower alkyl group having 1-4 carbon atoms, an alkoxy group having 1-4
carbon atoms, halogen, a nitro group or an amino group;
(c) acylating said ester 3 to an acyl derivative 4 of the general formula
##STR5##
wherein Y.sup.-, which is the same as or different than X.sup.-, is a
counterion imparting solubility to 4 and OR is a leaving group wherein R
is selected from an alkylsulfonyl having 1-12 carbon atoms, formyl and
trifluoroacetyl,
by reacting 3 with an acylating agent (1) of the formula RY wherein Y is a
halogen or (2) an anhydride precursor for R, and R has the above defined
meaning, with an organic base, in a basic solvent or in at least one inert
organic solvent, at 0.degree. C.-50.degree. C., for 1-24 hours;
(d) converting the COR.sub.1 group of said acylderivative 4 to a carboxylic
group, to obtain an acyl D-(+)-carnitine 5 of the formula
##STR6##
(e) lactonizing said acyl D-(+)-carnitine 5 to a lactone 6 of
L-(-)-carnitine of the formula
##STR7##
by treating 5 in a basic environment, and (f) converting said lactone 6 to
L-(-)-carnitine inner salt by treating 6 in a basic solution and isolating
said L-(-)-carnitine inner salt.
2. The process of claim 1, wherein step (d) comprises hydrogenating said
acylderivative 4 in an aqueous solution at pH 2-4, at 0.degree.
C.-25.degree. C., for 1-8 hours, at 1-4 hydrogen atmospheres, in the
presence of a hydrogenation catalyst.
3. The process of claim 1, wherein said steps (d), (e) and (f) are carried
out as a single step, without isolating said intermediate compounds 5 and
6.
4. The process of claim 1, wherein:
X is a halogen, phosphate, perchlorate, metaperiodate, tetraphenylborate,
or alkylsulfonate having 1-12 carbon atoms;
R.sub.1 is benzyloxy; and
R is methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),
p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl (nosyl),
trifluoromethanesulfonyl (triflyl), nonafluoromethanesulfonyl.(nonaflyl)
or 2,2,2-trifluoroethansulfonyl (tresyl).
5. A process for producing L-(-)-carnitine comprising
(a) esterifying D-(+)-carnitine to an ester 3 of the general formula
##STR8##
wherein R.sub.1 is (i) a straight or branched alkoxy group having 1-11
carbon atoms or (ii) an arylalkoxy or diarylalkoxy group wherein the aryl
group is a monocyclic or bicyclic aryl group containing 5 to 12 carbon
atoms and the alkyl group has 1-4 carbon atoms, and wherein said
arylalkoxy or diarylalkoxy groups can be optionally substituted with a
lower alkyl group having 1-4 carbon atoms, an alkoxy group having 1-4
carbon atoms, halogen, a nitro group or an amino group;
(b) acylating said ester 3 to an acyl derivative 4 of the general formula
##STR9##
wherein Y.sup.-, which is the same as or different than X.sup.-, is a
counterion imparting solubility to 4 and OR is a leaving group wherein R
is selected from an alkylsulfonyl having 1-12 carbon atoms, formyl and
trifluoroacetyl,
by reacting 3 with an acylating agent of the formula RY wherein Y is a
halogen or RY is an anhydride and R has the above defined meaning, with an
organic base, in a basic solvent or in at least one inert organic solvent,
at 0.degree. C.-50.degree. C., for 1-24 hours;
(c) converting the COR.sub.1 group of said acylderivative 4 to a carboxylic
group, to obtain an acyl D-(+)-carnitine 5 of the formula
##STR10##
(d) lactonizing said acyl D-(+)-carnitine S to a lactone 6 of
L-(-)-carnitine of the formula
##STR11##
by treating 5 in a basic environment, and (e) converting said lactone 6 to
L-(-)-carnitine by treating 6 in a basic solution and isolating
L-(-)-carnitine inner salt.
6. The process of claim 5, wherein steps (c), (d), and (e) are carried out
as a single step, without isolating said intermediate compounds 5 and 6.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for manufacturing
L-(-)-carnitine from a starting compound containing an asymmetrical carbon
atom having a configuration opposite to that of L-(-)-carnitine. The
process of the present invention overcomes the drawbacks of conventional
processes which first convert a starting compound into an achiral
intermediate, generally crotonobetaine or gamma-butyrobetaine, and then
convert the achiral intermediate to L-(-)-carnitine. The process of the
present invention uses D-(+)-carnitine or a derivative thereof as a
starting compound.
2. Discussion of the Background
Carnitine contains a single center of asymmetry and therefore exists as two
enantiomers, designated D-(+)-carnitine and L-(-)-carnitine. Of these,
only L-(-)-carnitine is found in living organisms, where it functions as a
vehicle for transporting fatty acids across mitochondrial membranes.
Whilst L-(-)-carnitine is the physiologically-active enantiomer, racemic
D,L-carnitine has conventionally been used as a therapeutic agent. It is
now recognized, however, that D-(+)-carnitine is a competitive inhibitor
of carnitine acyltransferases, and that it diminishes the level of
L-(-)-carnitine in myocardium and skeletal muscle.
It is therefore essential that only L-(-)-carnitine be administered to
patients undergoing haemodialysis treatment or treatment for cardiac or
lipid metabolism disorders. The same requirement applies to the
therapeutic utilization of acyl derivatives of carnitine for treating
disorders of the cerebral metabolism, peripheral neuropathies, peripheral
vascular diseases and the like. These disorders are typically treated with
acetyl L-(-)-carnitine and propionyl L-(-)-carnitine, which are obtained
by acylating L-(-)-carnitine.
Various chemical procedures have been proposed for the industrial-scale
production of carnitine. Unfortunately, these procedures are not
stereospecific and produce racemic mixtures of D-(+)- and L-(-)-isomers.
It is thus necessary to apply resolution methods in order to separate the
enantiomeric constituents of the racemate.
Typically, the D,L-racemic mixture is reacted with an optically active acid
(e.g. D-(-)-tartaric acid, D-(+)-camphorsulfonic acid,
(+)-dibenzoyl-D-(-)-tartaric acid, N-acetyl-L-(+)-glutamic acid and
D-(+)-camphoric acid) to obtain two diastereoisomers which can be
separated from each other. In the classic process disclosed in U.S. Pat.
No. 4,254,053, D-(+)-camphoric acid is used as the resolution agent of a
racemic mixture of D,L-carnitinamide, obtaining D-(+)-carnitinamide as a
by-product, and L-(-)-carnitinamide which, by hydrolysis, gives
L-(-)-carnitine.
However, these resolution procedures are complex and costly, and in all
cases result in the production of equimolar quantities of L-(-)-carnitine
and D-(+)-carnitine or a precursor thereof as by-product, having
configuration opposite to that of L-(-)-carnitine. Several microbiological
processes have recently been proposed for producing L-(-)-carnitine via
stereospecific transformation of achiral derivatives obtained from the
huge amounts of D-(+)-carnitine (or of a precursor thereof, such as
D-(+)-carnitinamide) which are generated as by-products in the industrial
production of L-(-)-carnitine.
These processes are generally predicated upon the stereospecific hydration
of crotonobetaine to L-(-)-carnitine, and differ principally by virtue of
the particular microorganism employed to accomplish the biotransformation
of interest. See, for example, the processes disclosed in: EP 0 12 1444
(HAMARI), EP 0 122 794 (AJINOMOTO), EP 0 148 132 (SIGMA-TAU), JP 275689/87
(BIORU), JP 61067494 (SEITETSU), JP 61234794 (SEITETSU), JP 61234788
(SEITETSU), JP 61271996 (SEITETSU), JP 61271995 (SEITETSU), EP 0 410 430
(LONZA), EP 0 195 944 (LONZA), EP 0 158 194 (LONZA), and EP 0 457 735
(SIGMA-TAU).
On the other hand, JP 62044189 (SEITETSU) discloses a process for
stereoselectively producing L-(-)-carnitine starting from
gamma-butyrobetaine, which is in turn obtained enzymically from
crotonobetaine.
All of these processes have several drawbacks. First, D-(+)-carnitine must
first be converted to an achiral compound (crotonobetaine,
gamma-butyrobetaine) before it can be used as the starting compound in all
of the aforesaid microbiological processes.
In addition, the microbiological procedures proposed to date have not
proven practicable for manufacturing L-(-)-carnitine on an industrial
scale for one or more of the following reasons:
(i) the yield of L-(-)-carnitine is extremely low;
(ii) the microorganisms must be cultivated in a costly nutritive medium;
(iii) the microorganism can only tolerate low concentrations [up to 2-3%
(w/v)] of crotonobetaine;
(iv) side reactions occur, such as the reduction of crotonobetaine to
gamma-butyrobetaine or the oxidation of L-(-)-carnitine to
3-dehydrocarnitine. These side reactions reduce the final yield of
L-(-)-carnitine.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide an efficient
method for producing L-(-)-carnitine from a derivative of D-(+)-carnitine.
The process of the present invention overcomes all of the aforesaid
drawbacks of the known processes, allowing high yields of L-(-)-carnitine
to be obtained starting from a by-product having configuration opposite to
that of L-(-)-carnitine with no need to first convert the starting
by-product into an achiral intermediate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The process of the invention is illustrated in the following reaction
scheme:
##STR1##
With reference to the reaction scheme, the D-(+)-carnitinamide salt 1,
wherein X is any suitable counterion is hydrolyzed to D-(+)-carnitine 2
via conventional procedures (see, for example JP 287065/1989, incorporated
herein by reference). X is suitably a halogen, preferably chloride;
phosphate; perchlorate; metaperiodate; tetraphenylborate; an
alkylsulfonate having 1-12 carbon atoms, preferably dodecylsulphonate;
trifluoroacetate; tetrahalogenborate; fumarate or an alkylsulphate having
10-14 carbon atoms.
D-(+)-carnitine 2 is then converted to the ester 3 in order to protect the
carboxyl group. Suitable esters 3 are those wherein R.sub.1 is (1) a
straight or branched alkoxy group having 1-11 carbon atoms or (2) an
arylalkoxy or diarylalkoxy group wherein the aryl is a monocyclic or
bicyclic aryl and the alkyl has 1-4 carbon atoms. Suitable monocyclic or
bicyclic aryl groups contain 5-12 carbon atoms and can be optionally
substituted with a lower alkyl group having 1-4 carbon atoms; an alkoxy
group having 1-4 carbon atoms; halogen, preferably fluorine or chlorine; a
nitro group or an amino group. Suitable arylalkoxy or diarylalkoxy groups
include p-methoxybenzyloxy, 1-naphthalenemethoxy, 2-naphthalenemethoxy,
and diphenylmethoxy. A particularly preferred arylalkoxy group is
benzyloxy.
The esterification of 2 to 3 is carried out via conventional procedures.
For instance, when R.sub.1 is benzyloxy, the preparation of
D-(+)-carnitine benzyl ester is carried out as disclosed in Biochim.
Biophys. Acta (1967) 137:98, incorporated herein by reference.
The ester 3 is then converted to the acyl derivative 4. Y, which can be the
same as X, is preferably a counterion imparting solubility to 4. OR is a
leaving group wherein R is an alkylsulfonyl group having 1-12 carbon
atoms, formyl or trifluoroacetyl. Preferably, the alkylsulfonyl group is
selected from methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),
p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl (nosyl),
trifluoromethanesulfonyl (triflyl), nonafluoromethanesulfonyl (nonaflyl)
and 2,2,2-trifluoroethanesulfonyl (tresyl). Mesyl is particularly
preferred.
The acylation of 3 to 4 is carried out by reacting the ester 3 with an
acylating agent RY wherein Y is halogen, or RY itself is an anhydride and
R is an acyl group as defined above. Preferably RY is the chloride of the
selected acyl group.
The acylation reaction is suitably carried out in pyridine, alkylpyridines,
or other basic solvents such as triethylamine or in a mixture of an
anhydrous, inert organic solvent such as acetonitrile or methylene
chloride with a base such as pyridine, lutidine, picoline or
polyvinylpyridine.
The acylating agent is suitably added at ratios ranging from 1:1 to 1:10,
preferably 1:3. The resulting reaction mixture is kept under stirring at
temperatures comprised between 0.degree. C. and 50.degree. C. for 1-24
hours Compound 4 is isolated by precipitation with a suitable solvent such
as ethyl ether or hexane and purified by dissolving it in water and
extracting with an organic solvent.
The carboxyl group is restored into compound 4 via known procedures, to
yield acyl D-(+)-carnitine 5. In some instances, if needed, compound 4 is
subjected to hydrogenation.
Hydrogenation of 4 is suitably carried out in an aqueous solution, at pH
2-4, or in methanol at 0.degree. C.-25.degree. C., for 1-8 hours, at 1-4
hydrogen atmospheres, in the presence of a hydrogenation catalyst such as
5% or 10% Pd/C. Acyl D-(+)-carnitine 5 can be isolated by filtering off
the catalyst and lyophilizing or concentrating the aqueous solution.
Acyl D-(+)-carnitine 5 is then converted to the lactone 6 of
L-(-)-carnitine. The lactonization is suitably carried out in an aqueous
basic environment: either with NaHCO.sub.3 (ratio 1:1) or with an
AMBERLITE IRA-402 (manufactured by Rohm & Haas Co., GERMANY) basic resin
activated in HCO.sub.3.sup.- form or with an LA2 resin (Rohm & Haas). The
lactone is isolated by evaporating the aqueous solution or precipitating
it as a salt (for example, as tetraphenylborate or reineckate).
Finally, lactone 6 is suitably converted to L-(-)-carnitine inner salt 7.
The lactone is dissolved in water and the resulting solution treated with
a base such as NaHCO.sub.3 (ratio 1:1), for 8-24 hours.
L-(-)-carnitine can suitably be purified from the salts which are formed
from the X.sup.- anion, from the excess, if any, of the acyl halogenide,
from pyridine, and the like, by chromatographing the aqueous solution on a
strongly acidic resin such as IR 120 (Rohm & Haas), eluting with water and
then with NH.sub.4 OH, or alternatively eluting first on a strongly basic
resin such as AMBERLITE IRA 402 (Rohm & Haas) activated in OH form and
thereafter on a weakly acid resin such as AMBERLITE IRC-50 (Rohm & Haas).
It should be understood that, whereas the process disclosed above has been
described, for the sake of clarity, as a sequence of six distinct
operating steps, the corresponding industrial process consists of four
steps only. When the process of the present invention is carried out as an
industrial process, the acyl D-(+)-carnitine ester 4 can be directly
converted to L-(-)-carnitine inner salt 7 without isolating either the
acyl D-(+)-carnitine 5 or the lactone 6.
In fact, the ester of acyl D-(+)-carnitine 4 is hydrogenated and the
hydrogenation catalyst filtered off. The resulting aqueous solution is
brought to pH 7-9, preferably 8-9 and kept at this pH value for 30-50
hours yielding L-(-)-carnitine. L-(-)-carnitine thus obtained is purified
by removing the salts by treatment with acidic and basic resins.
In the following example which describes one embodiment of the process of
the invention, the intermediate compounds 4, 5 and 6 were isolated so as
to exhaustively characterize them from a physico-chemical standpoint,
insofar as these intermediates are novel compounds.
It will be, however, apparent to any expert in organic synthesis that the
industrial process comprises the following steps only:
(a) hydrolysis of D-(+)-carnitinamide 1 to D-(+)-carnitine 2;
(b) esterification of D-(+)-carnitine 2 to the ester 3 to protect the
carboxyl group;
(c) acylation of the hydroxyl group of ester 3 with an acylating agent RY
wherein Y is a halogen or RY itself an anhydride, with the resulting
formation of a leaving group OR wherein R has the previously defined
meanings, thus obtaining the ester 4 of D-(+)-carnitine; and
(d) conversion of 4 to L-(-)-carnitine inner salt 7.
Having generally described this invention, a further understanding can be
obtained by reference to certain specific examples which are provided
herein for purposes of illustration only and are not intended to be
limiting unless otherwise specified.
In the following examples, the conversion of D-(+)-carnitinamide to
D-(+)-carnitine and the conversion of the latter compound to ester 3 are
not described for the sake of brevity and since those conversions can be
carried out via procedures well-known to any expert in organic synthesis.
Moreover, with reference to the numbering of the compound show in the
reaction scheme, the lower-case letters "a", "b" and "c" are used in the
example to indicate X.sup.- =perchlorate, chloride and methanesulfonate,
respectively.
EXAMPLE
Preparation of Methanesulfonyl D-(+)-carnitine Benzyl Ester Perchlorate
(4a)
Methanesulfonyl chloride (25.77 g; 225 mmoles) was added in the space of
five minutes to a solution of D-(+)-carnitine benzylester perchlorate
(24.4 g; 75 mmoles) in anhydrous pyridine (100 mL) cooled in an ice bath.
At the end of the addition, the solution was kept under stirring at room
temperature for 1 hour and 45 minutes. The solution was then poured into
an Erlenmeyer flask containing 500 mL Et.sub.2 O under stirring.
The oily precipitate obtained by decantation of Et.sub.2 O was taken up
with CH.sub.2 Cl.sub.2 (300 mL), that solution was washed with 2N HCl
(4.times.5 mL), saturated solution of NaCl (1.times.20 mL) and dried over
anhydrous Na.sub.2 SO.sub.4.
Following evaporation of the organic phase, 22 g of an amorphous solid were
obtained. Yield 70%. Differential thermal analysis: it decomposes at about
180.degree. C. [.alpha.].sub.D.sup.25 =+20.0.degree. [c=1% MeOH)
______________________________________
TLC = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/28/7/10.5/10.5
Rf = 0.5
______________________________________
Elementary analysis for C.sub.15 H.sub.24 ClNO.sub.9 S
C % H % N % Cl %
______________________________________
Calculated 41.91 5.63 3.25 8.25
Found 41.81 4.72 3.28 8.10
______________________________________
.sup.1 H NMR ((CD.sub.3).sub.2 CO): .delta. 7.45-7.30 (m, 5H, aromatics);
5.71-5.62 (m, 1H, --CHOMs); 5.20 (s, 2H, --CH.sub.2 Ph); 4.24-4.02 (m, 2H,
--CH.sub.2 N.sup.+ Me.sub.3); 3.47 (s, 9H, --N.sup.+ Me.sub.3); 3.30 (s,
3H, CH.sub.3 SO.sub.3 --) 3.20 (2H, d, --CH.sub.2 COO.sup.-) .sup.13 C NMR
((CD.sub.3).sub.2 CO): .delta. 169.413; 136.685; 129.153; 71.902 67.496;
54.683; 39.387; 38.640 IR (KBr)=.upsilon.(cm.sup.-1) 1735 (--C.dbd.O),
1341 and 1174 (CH.sub.3 SO.sub.3 --)
HPLC
Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant=CH.sub.3 CN/KH.sub.2 PO.sub.4 50 mM (65/35) pH=3.5 with H.sub.3
PO.sub.4
Flow rate=0.75 ml/min
Retention time=9.35 min
Detector=RI Waters 410
Preparation of Methanesulfonyl D-(+)-carnitine Benzyl Ester Chloride (4b)
18.3 g (42.6 mmoles) of methanesulfonyl D-(+)-carnitine benzyl ester
perchlorate were dissolved in 300 mL CH.sub.3 OH and few mL CH.sub.3 CN
(till complete dissolution). The solution thus obtained was percolated
through AMBERLYST A-21 resin (300 g) activated by percolating therethrough
1N HCl, then H.sub.2 O till neutrality and finally CH.sub.3 OH. Following
methanol evaporation, 15.5 of a solid product were obtained. Yield:
quantitative. Differential thermal analysis: it decomposes at about
150.degree. C. [.alpha.].sub.D.sup.25 =+22.6.degree. [c=1% MeOH)
______________________________________
TLC = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/28/7/10.5/10.5
Rf = 0.5
______________________________________
Elementary analysis for C.sub.15 H.sub.24 ClNO.sub.5 S
C % H % N % Cl %
______________________________________
Calculated (+3.3% di.H.sub.2 O)
47.62 6.76 3.70 9.37
Found 47.88 7.52 3.77 9.04
______________________________________
.sup.1 H NMR (D.sub.2 O): .delta. 7.50-7.45 (m, 5H, aromatics); 5.70-5.62
(m, 1H, --CHOMs); 5.40-5.30 (m, 2H, --CH.sub.2 Ph); 4.03-3.72 (m, 2H,
--CH.sub.2 N.sup.+ Me.sub.3); 3.25 (s, 3H, CH.sub.3 SO.sub.3 --) 3.22 (s,
9H --N.sup.+ Me.sub.3); 3.15 (2H, d, --CH.sub.2 COO.sup.-) .sup.13 C NMR
(D.sub.2 O): .delta. 172.789; 137.950; 131.695; 73.929; 70.651; 56.831;
41.475; 40.920 IR (pure)=.upsilon.(cm.sup.-1) 1734 (--C.dbd.O), 1340 and
1174 (CH.sub.3 SO.sub.3.sup.-)
HPLC
Column Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant CH.sub.3 CN/KH.sub.2 PO.sub.4 50 mM (65/35) pH=3.5 with H.sub.3
PO.sub.4
Flow rate 0.75 ml/min
Retention time=9.41 min
Detector=RI Waters 410
Preparation of Methanesulfonyl D-(+)-carnitine Perchlorate (5a)
10% Pd/C (300 mg) was added to a solution of methanesulfonyl
D-(+)-carnitine benzyl ester perchlorate (3.0 g; 7 mmoles) in CH.sub.3 OH
(50 mL).
The resulting mixture was kept under stirring in a hydrogen atmosphere at
45 p.s.i. (219.7 kg/m.sup.2) in a Parr apparatus for 4 hours. After the
catalyst was filtered off and the solvent evaporated, 2.3 g of a white
solid product were obtained. Yield: quantitative. Differential thermal
analysis: incipient decomposition at about 170.degree. C.
[.alpha.].sub.D.sup.25 =+19.6.degree. [c=1% MeOH)
______________________________________
TLC = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/20/7/10.5/10.5
Rf = 0.15
______________________________________
Elementary analysis for C.sub.8 H.sub.18 ClNO.sub.9 S
C % H % N % Cl %
______________________________________
Calculated 28.28 5.34 4.12 10.43
Found 28.78 5.34 4.15 10.23
______________________________________
.sup.1 H NMR (D.sub.2 O): .delta. 5.68-5.59 (m, 1H, --CHOMs, ); 4.05-3.75
(m, 2H, --CH.sub.2 N.sup.+ Me.sub.3); 3.33 (s, 3H, CH.sub.3 SO.sub.3 --)
3.27 (s, 9H-N.sup.+ Me.sub.3); 3.15-3.00 (m, 2H, --CH.sub.2 COOH) .sup.13
C NMR (D.sub.2 O): .delta. 175.192; 74.423; 70.838; 56.971; 41.662; 40.774
IR (KBr)=.upsilon.(cm.sup.-1) 1731 (C.dbd.O), 1340 and 1174 (CH.sub.3
SO.sub.3 --)
HPLC
Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant=CH.sub.3 CN/KH.sub.2 PO.sub.4 50 mM (65/35) pH=3.5 with H.sub.3
PO.sub.4
Flow rate=0.75 ml/min
Retention time=11.33 min
Detector=RI Waters 410
Preparation of Methanesulfonyl D-(+)-carnitine Chloride (5b)
10% Pd/C (500 mg) was added to a solution of methanesulfonyl
D-(+)-carnitine benzyl ester chloride (5.1 g; 13.9 mmoles) in H.sub.2 O
(60 mL) acidified to pH 4 with 1N HCl. The resulting mixture was kept
under stirring in a hydrogen atmosphere, at 45 p.s.i. (219.7 kg/m.sup.2)
in a Parr apparatus for 4 hours.
The catalyst was filtered off and the aqueous solution lyophilized, giving
3.8 g of a white solid product. Yield: quantitative. Differential thermal
analysis: it decomposes at about 150.degree. C. [.alpha.].sub.D.sup.25
=+29.5.degree. [c=1% H.sub.2 O)
______________________________________
TCL = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/20/7/10.5/10.5
Rf = 0.15
______________________________________
Elementary analysis for C.sub.8 H.sub.18 ClNO.sub.5 S
C % H % N % Cl %
______________________________________
Calculated 34.84 6.58 5.10 12.86
Found 35.37 6.82 5.24 12.45
______________________________________
.sup.1 H NMR (D.sub.2 O): .delta. 5.70-5.60 (m, 1H, --CHOMs, ); 4.06-3.75
(m, 1H, --CH.sub.2 N.sup.+ Me.sub.3); 3.33 (s, 3H, CH.sub.3
SO.sub.3.sup.-) 3.27 (s, 9H --N.sup.+ Me.sub.3); 3.15-3.00 (m, 2H,
--CH.sub.2 COOH) .sup.13 C NMR (D.sub.2 O): .delta. 175.326; 74.530;
70.851; 56.964; 41.668; 40.914 IR (KBr)=.upsilon.(cm.sup.-1) 1720
(C.dbd.O), 1335 and 1175 (CH.sub.3 SO.sub.3 --)
HPLC
Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant=CH.sub.3 CN/KH.sub.2 PO.sub.4 50 mM (65/35) pH =3.5 with H.sub.3
PO.sub.4
Flow rate=0.75 ml/min
Retention time=11.38 min
Detector=RI Waters 410
Preparation of the Lactone of L-(-)-carnitine Chloride (6b)
NaHCO.sub.3 (0.46 g; 5.4 mmoles) was added to a solution of methanesulfonyl
D-(+)-carnitine chloride (1.5 g; 5.4 mmoles) in H.sub.2 O (25 mL) and the
resulting solution was kept under stirring for 20 hours. The solution was
then lyophilized, the residue taken up with CH.sub.3 CN and the
undissolved solid filtered off. Following solvent evaporation, 0.98 g, of
the title compound were obtained. Yield: quantitative.
______________________________________
TLC = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/28/7/10.5/10.5
Rf = 0.1
______________________________________
.sup.1 NMR (D.sub.2 O): .delta. 5.33-5.24 (m, 1H, --CHOCO--); 3.96-3.88 (m,
3H), --CH.sub.2 N.sup.+ Me.sub.3, --CHHCO--); 3.53-3.44 (m, 1H,
--CHHCOO--); 3.24 (s, 9H, --N.sup.+ Me.sub.3) .sup.13 C NMR (D.sub.2 O):
.delta. 172.428; 70.671; 68.094; 56.991; 41.394 IR
(KBr)=.upsilon.(cm.sup.-1) 1850 (C.dbd.O)
HPLC
Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant=CH.sub.3 CN/KH.sub.2 PO.sub.4 50 mM (65/35) pH=3.5 with H3PO.sub.4
Flow rate=0.75 ml/min
Retention time=19.23 min
Detector=RI Waters 410
Preparation of the Lactone of L-(-)-carnitine Methanesulfonate (6c)
An aqueous solution of methanesulfonyl D-(+)-carnitine chloride (1.5 g; 5.4
mmoles) was perchlorated through an IRA-402 resin (30 g) activated to
HCO.sub.3.sup.- form and cooled to 5.degree. C., eluting with water at
5.degree. C. till complete elution (controlled by TCL). The eluate was
kept at room temperature for 4 hours. Following evaporation of the aqueous
solution, 1.3 g of a raw product which was taken up with CH.sub.3 CN, were
obtained. Evaporation of the organic solvent yielded 1 g of a white solid.
Yield: 80% Differential thermal analysis: incipient decomposition at
160.degree. C. [.alpha.].sub.D.sup.25 =+24.7.degree. (c=1% MeOH)
______________________________________
TCL = silica gel
Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH
42/28/7/10.5/10.5
Rf = 0.1
______________________________________
Elementary analysis for C.sub.8 H.sub.17 NO.sub.5 S
C % H % N %
______________________________________
Calculated 40.16 7.16 5.85
Found 39.61 7.13 5.77
______________________________________
.sup.1 H NMR (D.sub.2 O): .delta. 5.35-5.25 (m, 1H, --CHOCO--); 3.98-3.89
(m, 3H, --CH.sub.2 N.sup.+ Me.sub.3, --CHHCOO--), 3.54-3.46 (m, 1H,
--CHHCOO--); 3.26 (s, 9H, --N.sup.+ Me.sub.3); 2.81 (s, 3H, CH.sub.3
SO.sub.3 --) .sup.13 C NMR (D.sub.2 O): .delta. 172.428; 70.671; 68.094;
56.991; 45.320; 41.394 IR (KBr)=.upsilon.(cm.sup.-1) 1835 (C.dbd.O)
HPLC
Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm
Eluant=CH.sub.3 CN/KH.sub.3 PO.sub.4 50 mM (65/35) pH=3.5 with H.sub.3
PO.sub.4
Flow rate=0.75 ml/min
Retention time=19.48 min
Detector=RI Waters 410
Preparation of L-carnitine Inner Salt (7) from the Lactone of
L-(-)-carnitine Methanesulfonate (6c)
NaHCO.sub.3 (0.34 g; 4 mmoles) was added to a solution of the lactone of
L-(-)-carnitine methanesulfonate (0.96 g; 4 mmoles) in H.sub.2 O (20 mL)
and the resulting solution was kept under stirring at room temperature for
20 hours. The solution was then percolated through AMBERLITE IR-120 resin
(20 g) eluting first with water till neutrality to remove methanesulfonic
acid and then with 2% NH.sub.3 aqueous solution collecting the eluate till
complete elution of L-(-)-carnitine inner salt (controlled by TLC).
Following evaporation of the aqueous solution, 0.64 g of L-(-)-carnitine
inner salt were obtained.
Alternatively, the reaction mixture was percolated through IRA-402 resin
(20 g) activated to OH.sup.- form, eluting with H.sub.2 O till neutrality.
The eluate was then percolated through IRC-50 resin (20 g) till complete
elution of L-carnitine inner salt (controlled by TLC). Following
evaporation of the aqueous solution, 0.64 g of L-(-)-carnitine inner salt
were obtained.
Yield: quantitative
The enantiomeric excess (e.e.) was assessed via the following HPLC method,
after L-(-)-carnitine was derivatized with a chiral reagent. As chiral
reagent, (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC) was used.
column: Nova-pak C.sub.18 (4.mu.) Cartridge
length: 100 mm
diameter: 5.0 mm
Eluant:
Solution A: 5mM tetrabutylammonium hydroxide (TBA.sup.+ OH.sup.-), 50 mM
KH.sub.2 PO.sub.4 75 mL Acetonitrile 25 mL brought to pH 7 with 1N KOH
Solution B: Acetonitrile 75 mL 5 mM KH.sub.2 PO.sub.4 25 mL
______________________________________
Elution schedule
Time % A % B
______________________________________
0 100 0
15 100 0
16 0 100
22 0 100
23 100 0
30 stop
______________________________________
Detector = Perkin-Elmer Fluorimeter
Excitation = 260 nm
Slit = 10 nm
Emission = 315 nm
Slit = 5 nm
______________________________________
L-(-)-carnitine had previously been derivatized with FLEC via the following
method:
50 .mu.L of L-(-)-carnitine solution (prepared by dissolving 10 mg
carnitine in 50 mL of 50 mM TBA.sup.+OH.sup.- brought to pH 7 with
concentrated H.sub.3 PO.sub.4) and 200 .mu.L of solution consisting of 1
mL FLEC in 3 mL acetone, were kept under stirring at 80.degree. C. for 20
minutes.
The solution was cooled and 4 mL of solution A were added thereto, 5 .mu.L
of the resulting solution were injected L-(-)-carnitine K.sup.1 =5.79
D-(+)-carnitine K.sup.1 =4.82, absent
##EQU1##
Preparation of L-carnitine Inner Salt (7) from Methanesulfonyl-D-carnitine
Chloride (5b)
NaHCO.sub.3 (0.46 g; 5.4 mmoles) was added to a solution of
methanesulfonyl-D-carnitine chloride (1.5 g; 5.4 mmoles) in H.sub.2 O (25
mL) and the resulting solution was kept under stirring at room temperature
for 20 hours. Further NaHCO.sub.3 (0.46; 5.4 mmoles) was then added and
the solution was kept under stirring at room temperature for further 20
hours. The title compound was isolated as previously described for the
isolation of 7 from 6 b.
L-carnitine is obtained from methanesulfonyl-D-carnitine through the
formation of the lactone 6, as evidenced by NMR, HPLC, IR and TLC analysis
carried out on a sample obtained by lyophilizing a portion of the solution
20 hours following first NaHCO.sub.3 addition.
Having now fully described the invention, it will be apparent to one of
ordinary skill in the art that many changes and modifications can be made
thereto without departing from the spirit or scope of the invention as set
forth herein.
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